| Bellamya is a member of Viviparidae, Mesogastropoda, Gastropoda, Mollusca. It is one of macrobenthos in freshwater ecosystem, and wildly distributed in various lakes of Asia, Africa and North America. As an important genus of freshwater gastropods, Bellamya plays a significant role in the ecosystem. Due to environmental deterioration in lakes and overexploitation of resource of Bellamya by human in the late years, germplasm resources of Bellamya was damaged to some extent and the number of species was in decline. The classification of Bellamya was mostly based on the shell characteristics. Because of the variability of shell morphology, synonyms and taxon confusion were often found in reports. In this study, the genetic diversity and taxonomy research were investigated on the basis of molecular genetics, combined with morphological study. It was essential to investigate the population genetic structure, which could provide molecular genetic basis for genetic resource conservation. The main results were as follows:1. The partial sequence of the mitochondrial (COI and16S rRNA) and nuclear (H3and28S rRNA) of Bellamya genus were compared to determine their phylogenetic relationships, including Bellamy species from India, Africa and China. The phylogenetic trees were reconstructed based on Bayesian Inference methods. Viviparus ater and V. contectus were used as outgroup, and the sampled8species(Bellamya aeruginosa, B. angularis, B. quadrata, B. dispiralis, B. purificata, B. turritus, B. lapillorum, B. lapided) and7species(Bellamya sp.) in China, and Bellamy species from India and Africa download from GeneBank were used as ingroup. The phylogenetic analysis revealed that the worldwide Bellamya species form into three major clades, Indian species-clade, African species-clade and Chinese species-clade. Chinese Bellamya was the basal among the three clades. The African species was close to Indian species. Most of the African Bellamya species formed into three or four major lake-clades. However, in China Bellamya species-clade, neither the same species formed a cluster, nor the species in the same lake formed a cluster, which indicated that there was no significant genetic differentiation among populations and species. There was no significant difference between intra and interspecific genetic distance, further illustrating that the interspecific genetic differentiation was very low.2. A microsatellite-enriched genomic library of B. aeruginosa was successfully constructed by using the method of FIASCO (Fast Isolation by AFLP Sequences Containing Repeat), and2566microsatellite sequences were captured and deposited in GeneBank (Genbank assession No:JN555759-JN556037, JX018213-JX020499).391pairs of SSR-primers were designed by Primer v5.0, and100pairs of them were synthesized.33pairs of SSR-primers were successful amplified and had high polymorphic information content value (PIC), which ranged from0.244to0.889(average0.617). Allele number per locus ranged from5to13(average8.863). The expected heterozygosity (HE) and observed heterozygosity (Ho) varied from0.347to0.950and0.071to0.913with an average of0.780and0.543, respectively.3. The genetic structure of5Bellamy a species (B. aeruginosa, B. purificata, B. quadrata, B. angularis, B. dispiralis) were analyzed using7microsatellite locus.The genetic diversity analysis showed that all the5species had high observed heterozygosities and expected heterozygosities, indicating high genetic diversity among populations. Heterozygote deficiency was indicated with a significant deviation from the from Hardy-Weinberg equilibrium observed over all populations, while average HO value was consistently lower than HE value. Heterozygote deficiency had close relation with the high degree of inbreeding in population.There was no significant genetic differentiation among populations for each of the5Bellamya species, though high genetic distance was found between a very few populations. Analysis of molecular variance (AMOVA) indicated that the genetic variance mainly presented in individuals within populations, there were no significant genetic differences among populations. Based on the molecular analysis, the investigated populations showed obvious gene flow among populations.AMOVA from microsatellite data indicated that no significant genetic structure of Bellamya existed at various hierarchical levels (among regions, among populations within regions, and within population). The proportions of genetic variance among regions were very low, indicating that populations had not form a pattern of obvious geographical distribution. The discriminant analysis of principal components (DAPC) method revealed no clear separation of regional clusters,12populations of B. aeruginosa were divided into five clusters,8populations of B. purificata were divided into four clusters, and7population of B. quadrata were divided into three clusters. However, the dominant populations in each cluster also couldn’t form obvious geographical distribution pattern. AMOVA revealed very low amount of genetic differentiation among the five species. The 34populations of the5species were divided into13clusters, and no species clade was found in these clusters.4. The genetic structure of B. aeruginosa, B. quadrat a and B. purificata were studied using mtDNA COI gene.The3Bellamya species showed high genetic diversity among populations.12populations of B. aeruginosa had116sequences, in which81haplotypes and167polymorphic sites were detected, and the haplotype and nucleotide diversity ranged from0.8670to0.9780and0.0130to0.0449, respectively.8populations of B. purificata had77sequences, in which66haplotypes and155polymorphic sites were detected, and the haplotype and nucleotide diversity ranged from0.8890to1.0000and0.0067to0.0416, respectively.7population of B. quadrata had63sequences, in which51haplotypes and158polymorphic sites were detected, and the haplotype and nucleotide diversity ranged from0.8210to1.0000and0.0313to0.0474, respectively.When all populations pooled in one group for each of the three species, All the Fu’s Fs value of the three species were significant negative, for B. aeruginosa Fs=-23.8828(P<0.001), for B. purificata Fs=-24.0457(P<0.001), for B. quadrata Fs=-12.9900(P=0.011). Besides, Mismatch distribution of pairwise nucleotide differences of mtDNA COI showed unimodal pattern for each of the three species. The results supported that for B. aeruginosa, B. purificata and B. quadrata all had ever experienced demographic expansion. Based on1.22%±0.27%per million years (Myr), the demographic expansion time for B. aeruginosa, B. purificata and B. quadrata was about2.197Ma,0.256Ma and2.217Ma respectively, which were in Pleistocene.AMOVA analysis indicated that there was no obvious genetic structure of the three species existed at various hierarchical levels (among regions, among populations within regions, and within population). The proportion of genetic variance among regions was negative, indicating that populations had not form a pattern of obvious geographical distribution. The network generated from haplotype data set showed that the haplotype of different populations linked together, and displayed no apparent lineage clades association with sampling locations. AMOVA also revealed very low amount of genetic differentiation among the three species,1.05%of the total variation was distributed among the three species, and genetic distance between the three species ranged from0.004to0.030.5. According to six morphological characters, principal component analysis and cluster analysis were used to investigate morphological variations of32populations of5Bellamya species (B. aeruginosa, B. purificata, B. quadrata, B. angularis, B. dispiralis). The result showed that there were different degrees of morphological variation among populations, but no obvious morphological differentiation was found in the same Bellamya species. Discriminant analysis also indicated that there were no significant differences among populations, with low disciminant accuracy in most populations. The disciminant accuracy was33.33%-66.67%for B. aeruginosa;45%-80%for B. purificata;33.33%-86.67%for B. quadrata;53.33%-93.33%for B. angularis,46.15%-76.19%for B. dispiralis. The results of principal component analysis and cluster analysis for32populations showed that the5species could be classified into three morphologically different groups. The first group just included B. aeruginosa, the second group included B. purificata and B. angularis, and the third group included B. quadrata and B. dispiralis.6. The radula of five Bellamya species (B. aeruginosa, B. purificata, B. angularis, B. quadrata and B. dispiralis) samping from the same lake was compared on the basis of scanning electron microscopic observation. Results showed that all of them have the same dentition formula2·1·1·1·2, consists of central teeth, lateral teeth, inner marginal teeth and outer marginal teeth. There was no remarkable interspecific morphological difference in the four parts of radula, but significant differences in the amount of the denticles on the central teeth, lateral teeth and inner marginal teeth among the five species.In conclusion, there was no obvious interpopulation and interspecific genetic differentiation for Bellamya in China. The phylogenetic inference and analysis of genetic structure could not support the taxonomic category of Bellamya in China. Morphological analysis indicated that there were differences among species, and the difference of radula structure was obvious. Consequently, the shell morphology characteristic and radula structure is still the most important basis for Bellamya classification, it is necessary to find better molecular marker to do research on the taxonomic category of Bellamya. |
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